Production of squalene using yeast

ABSTRACT

Provided herein compositions and methods for producing isoprenoids, including squalene. In certain aspects and embodiments provided are genetically altered yeast and uses therefore. In some aspects and embodiments, the genetically altered yeast produce isoprenoids, preferably squalene. The genetically altered yeast may have alterations in the expression or activity of enzymes involved in squalene production, for example, acetyl-CoA carboxylase (or “ACCase”), HMG-CoA reductase, squalene epoxidase, and squalene synthase. One or more genes of a genetically altered yeast may be modified by gene repair oligonucleobases. Also are provided methods of producing squalene using a genetically altered yeast. The invention also provides squalene produced by genetically altered yeast.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims priority to U.S. Provisional PatentApplication No. 61/055,931, entitled “Production of Squalene UsingYeast,” filed May 23, 2008, which is hereby incorporated by reference inits entirety for all purposes.

FIELD OF THE INVENTION

Provided are methods and compositions for producing isoprenoids such assqualene using yeast.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Isoprenoids, such as squalene, are commercially important types oflipids. They have excellent lubricity, oxidative stability, low pourpoints, low freezing points, high flash points, and facilebiodegradability. Squalene is currently produced by extraction fromolive oil or cold water shark liver oil at a high unit cost. Because ofthe high unit cost, economically feasible uses for squalene and squalane(the fully hydrogenated derivative of squalene) are in small marketapplications such as watch lubricants, pharmaceuticals/nutraceuticals,cosmetics, perfumes and as chemical intermediates for high-valueproducts.

There exist, however, significant potential markets for biodegradablelubricants, lubricant additives, and hydraulic fluids. Biodegradabilityof these products is particularly important for environmentallysensitive applications, such as agricultural applications, or whereconsiderable lubricant or hydraulic fluids may be lost to theenvironment. The potential markets for biodegradable lubricants,lubricant additives, and hydraulic fluids are quite large, estimated tobe on the order of five million metric tons per annum.

Biodegradable lubricants, lubricant additives, and hydraulic fluidsderived from vegetable and animal fats and oils are available, but theyhave drawbacks. They typically solidify at relatively high temperatures(i.e., they solidify in cold weather) and have flash points that are toolow for use in hot conditions, (i.e., they break down or combust undernormal hot engine conditions).

Thus, a cost effective method of production of squalene is desired thatwould allow for large-scale manufacturing and widespread use of squaleneand squalane in biodegradable lubricants, lubricant additives, andhydraulic fluids.

Chang et al., (Appl. Microbiol. Biotechnol., 2008, 78, 963-72) disclosesthe discovery of a wild type yeast, Pseudozyma sp. JCC207, that produces“a large amount of squalene and several polyunsaturated fatty acids.”Chang et al. describe isolating Pseudozyma sp. JCC207 from seawatercollected near Guam, USA, and are unsure whether Pseudozyma sp. JCC207is a new species or a variant of P. regulosa or P. aphidis. In thearticle, “the efficiency of squalene production [of Pseudozyma sp.JCC207] was investigated under different conditions.”

Dow AgroSciences LLC, Using Yeast Fermentation to Produce Cost-Effectiveand Biodegradable Lubricants,http://statusreports.atp.nist.gov/reports/95-01-0148PDF.pdf, disclosesthat “[t]he company proposed to use genetic engineering to alter themetabolic characteristics of an oleaginous (oily) yeast to increase theyeast's ability to produce isoprenes through biosynthesis.”Specifically, four enzymes were targeted: ACCase, hydroxymethylglutarylCoA reductase (HMGR), squalene synthetase, and squalene epoxidase.

U.S. Pat. No. 5,460,949 discloses “[a] method increasing theaccumulation of squalene and specific sterols in yeast.” In particular,it is disclosed that “[s]qualene and sterol accumulation is increased byincreasing the expression level of a gene encoding a polypeptide havingthe HMG-CoA reductase activity.”

SUMMARY OF THE INVENTION

The instant invention provides compositions and methods for producingsqualene from yeast.

In one aspect, a genetically altered yeast that produces isoprenoids isprovided. In certain embodiments, the genetically altered yeast producessqualene.

In a related aspect, provided is a genetically altered yeast, whereinthe yeast is genetically altered such that it produces increased levelsof squalene as compared to the corresponding native yeast. In certainembodiments of the above aspects of the invention, the geneticallymodified yeast expresses one or more modified enzymes having one or moremutations. In certain embodiments of the above aspects the expressionlevel of one or more enzymes in the genetically altered yeast isincreased or decreased relative to the corresponding native yeast. Inrelated embodiments, the genetically modified yeast expresses one ormore modified enzymes having one or more mutations and the expressionlevel of one or more enzymes in the genetically altered yeast isincreased or decreased relative to the corresponding native yeast. Incertain preferred embodiments a genetically altered yeast as providedherein is genetically altered by introducing a mutation into an enzymeusing a gene repair oligobase. In some embodiments a genetically alteredyeast as provided herein is genetically altered by introducing one ormore mutation at or around the translation start site of a gene encodingan enzyme to increase or decrease expression of the enzyme, for example,as described in U.S. patent application Ser. Nos. 10/411,969 and11/625,586. In certain embodiments, the enzyme modified in a geneticallyaltered yeast as provided herein includes one or more enzymes selectedfrom the group consisting of acetyl-CoA carboxylase (or “ACCase”),HMG-CoA reductase, squalene epoxidase, squalene synthase, and ATPcitrate lyase.

A nucleobase comprises a base, which is a purine, pyrimidine, or aderivative or analog thereof. Nucleosides are nucleobases that contain apentosefuranosyl moiety, e.g., an optionally substituted riboside or2′-deoxyriboside. Nucleosides can be linked by one of several linkagemoieties, which may or may not contain phosphorus. Nucleosides that arelinked by unsubstituted phosphodiester linkages are termed nucleotides.“Nucleobases” as used herein include peptide nucleobases, the subunitsof peptide nucleic acids, and morpholine nucleobases as well asnucleosides and nucleotides.

An oligonucleobase is a polymer of nucleobases, which polymer canhybridize by Watson-Crick base pairing to a DNA having the complementarysequence. An oligonucleobase chain has a single 5′ and 3′ terminus,which are the ultimate nucleobases of the polymer. A particularoligonucleobase chain can contain nucleobases of all types. Anoligonucleobase compound is a compound comprising one or moreoligonucleobase chains that are complementary and hybridized byWatson-Crick base pairing. Nucleobases are either deoxyribo-type orribo-type. Ribo-type nucleobases are pentosefuranosyl containingnucleobases wherein the 2′ carbon is a methylene substituted with ahydroxyl, alkyloxy or halogen. Deoxyribo-type nucleobases arenucleobases other than ribo-type nucleobases and include all nucleobasesthat do not contain a pentosefuranosyl moiety.

An oligonucleobase strand generically includes both oligonucleobasechains and segments or regions of oligonucleobase chains. Anoligonucleobase strand has a 3′ end and a 5′ end. When anoligonucleobase strand is coextensive with a chain, the 3′ and 5′ endsof the strand are also 3′ and 5′ termini of the chain.

The term “gene repair oligonucleobase” is used herein to denoteoligonucleobases, including mixed duplex oligonucleotides,non-nucleotide containing molecules, single strandedoligodeoxynucleotides and other gene repair molecules as described indetail below.

In some embodiments, a genetically altered yeast as provided herein isderived from an oleaginous yeast. In certain preferred embodiments, agenetically altered yeast as provided herein is derived from a yeastselected from the group consisting of Cryptococcus curvatus, Yarrowialipolytica, Rhodotorula glutinlus, and Rhorosporidium toruloides. Insome preferred embodiments, the genetically altered yeast is derivedfrom a yeast selected from the group consisting of Cryptococcuscurvatus, Yarrowia lipolytica, and Rhodotorula glutinus. In relatedembodiments, the genetically altered yeast is derived from a yeastselected from the group consisting of Cryptococcus curvatus, andRhodotorula glutinus. In certain preferred embodiments, the geneticallyaltered yeast is not derived from Yarrowia lipolytica.

In certain preferred embodiments, an enzyme that is modified in agenetically altered yeast as provided herein is acetyl-CoA carboxylase(or “ACCase”). In some preferred embodiments acetyl-CoA carboxylase in agenetically altered yeast is modified such that its activity and/orexpression is decreased relative to the corresponding native yeast; orsuch that the activity and/or expression is eliminated. In otherembodiments, the acetyl-CoA carboxylase may be modified so that itssubstrate selectivity is altered. In some preferred embodiments, thegenetically altered yeast is modified such that the activity and orexpression of acetyl-CoA carboxylase is reduced relative to thecorresponding native yeast but the activity is not eliminated. In somepreferred embodiments, the genetically altered yeast is modified suchthat the activity and/or expression of acetyl-CoA carboxylase in thegenetically altered yeast is reduced to about 90%; or about 80%; orabout 70%; or about 60%; or about 50%; or about 40%; or about 30%; orabout 20%; or about 10%; or about 5% of the activity and/or expressionof the corresponding native yeast. In related embodiments, thegenetically altered yeast is modified such that the activity and/orexpression of acetyl-CoA carboxylase in the genetically altered yeast isbetween 90-95%; or 80-90%; or 70-80%; 60-70%; or 50-60%; or 40-50%; orabout 30-40%; or about 20-30%; about 10-20%; or about 5-10%; or about2-5% of the activity and/or expression of the corresponding nativeyeast.

In certain preferred embodiments, an enzyme that is modified in agenetically altered yeast as provided herein is HMG-CoA reductase. Insome preferred embodiments HMG-CoA reductase in a genetically alteredyeast is modified such that its activity and/or expression is increasedrelative to the corresponding native yeast. In other embodiments, theHMG-CoA reductase may be modified so that it substrate selectivity isaltered. In certain preferred embodiments, the genetically altered yeastis modified such that the activity and/or expression of HMG-CoAreductase in the genetically altered yeast is increased to at least1.2-fold; or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or10-fold; or 10-fold; or 20-fold; or 50-fold; or 100-fold; or 1000-fold;or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than theactivity and/or expression of the corresponding native yeast.

In certain preferred embodiments, an enzyme that is modified in agenetically altered yeast as provided herein is squalene epoxidase. Insome preferred embodiments squalene epoxidase in a genetically alteredyeast is modified such that its activity and/or expression is decreasedrelative to the corresponding native yeast; or such that the activityand/or expression is eliminated. In other embodiments, the squaleneepoxidase may be modified so that its substrate selectivity is altered.In some preferred embodiments, the genetically altered yeast is modifiedsuch that the activity and/or expression of squalene epoxidase isreduced relative to the corresponding native yeast but the activity isnot eliminated. In some preferred embodiments, the genetically alteredyeast is modified such that the activity and, or expression of squaleneepoxidase in the genetically altered yeast is reduced to about 90%; orabout 80%; or about 70%; or about 60%; or about 50%; or about 40%; orabout 30%; or about 20%; or about 10%; or about 5% of the activityand/or expression of the corresponding native yeast. In relatedembodiments, the genetically altered yeast is modified such that theactivity and/or expression of squalene epoxidase in the geneticallyaltered yeast is between 90-95%; or 80-90%; or 70-80%; 60-70%; or50-60%; or 40-50%; or about 30-40%; or about 20-30%O; about 10-20%; orabout 5-10%; or about 2-5% of the activity and/or expression of thecorresponding native yeast.

In certain preferred embodiments, an enzyme that is modified in agenetically altered yeast as provided herein is squalene synthase. Insome preferred embodiments squalene synthase in a genetically alteredyeast is modified such that its activity and/or expression is increasedrelative to the corresponding native yeast. In other embodiments, thesqualene synthase may be modified so that it substrate selectivity isaltered. In certain preferred embodiments, the genetically altered yeastis modified such that the activity and/or expression of squalenesynthase in the genetically altered yeast is increased to at least1.2-fold; or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or10-fold; or 10-fold; or 20-fold; or 50-fold; or 100-fold; or 1,000-fold;or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than theactivity and/or expression of the corresponding native yeast.

In certain preferred embodiments, an enzyme that is modified in agenetically altered yeast as provided herein is ATP citrate lyase. Insome embodiments, either or both subunits of ATP citrate lyase genes(for example, Yarrowia lipolytica ATP citrate lyase; GenoleveresYALI0D24431 g and YALI0E34793g) are modified as described herein. Incertain embodiments the activity of ATP citrate lyase in a modifiedyeast is increased by the insertion and/or heterologous expression of ananimal ATP lyase gene which comprises a single subunit holoenzyme. Insome preferred embodiments ATP citrate lyase in a genetically alteredyeast is modified such that its activity and/or expression is increasedrelative to the corresponding native yeast. In certain preferredembodiments, the genetically altered yeast is modified such that theactivity and/or expression of ATP citrate lyase in the geneticallyaltered yeast is increased to at least 1.2-fold; or 1.5-fold; or 2-fold;or 3-fold; or 4-fold; or 5-fold; or 10-fold; or 10-fold; or 20-fold; or50-fold; or 100-fold; or 1,000-fold; or 10,000-fold, or 100,000-fold; or1,000,000-fold higher than the activity and/or expression of thecorresponding native yeast.

In certain preferred embodiments of the above aspects, the geneticallyaltered yeast is a genetically modified yeast; in other preferredembodiments, the genetically modified yeast is a transgenic yeast.Further embodiments are a yeast that includes both transgenic andgenetic alterations.

The phrase “genetically modified yeast” as used herein refers to atransgenic yeast or a genetically altered yeast.

The term “native yeast” as used herein refers to a yeast that is notgenetically modified (i.e., transgenic or genetically altered). Nativeyeasts include wild type yeasts as well as yeasts that have beenselectively bred to attain particular characteristics.

The phrase “transgenic yeast” refers to a yeast having a gene fromanother yeast species or non-yeast species. Such a gene may be referredto as a “transgene.”

As used herein the term “target gene” refers to the gene encoding theenzyme to be modified.

The phrase “genetically altered yeast” refers to a yeast having one ormore genetic modifications, such as transgenes and/or modified enzymeswhich contain one or more designed mutation(s). Such designed mutationsmay result in a modified enzyme having an activity that is differentfrom the native enzyme. Such differences can include differences insubstrate specificity or level of activity. As used herein, a“transgenic yeast” is one type of a “genetically altered yeast”.

The phrase “oleaginous yeast” refers to a yeast that contains at leastabout 20% cell dry weight (cdw) lipid extractable from the organism. Thecapacity to accumulate levels of lipid at least about 20% cdw is notconfined to a particular genus; greater than about 20% cdw lipid hasbeen reported in Lipomyces lipofer, L. starkeyi, L. tetrasporus, Candidalipolytica, C. diddensiae, C. paralipolytica, C. curvata, Cryptococcusalbidus, Cryptococcus laurentii, Geotrichum candidum, Rhodotorulagraminus, Trichosporon pullulans, Rhodosporidium toruloides, Rhodotorulaglutinus, Rhodotorula gracilis, and Yarrowia lipolytica. See, e.g.,Tatsumi. et al. U.S. Pat. No. 4,032,405, and Rattray, Microbial Lipids,Vol. 1 (1998).

The term “about” as used herein means in quantitative terms plus orminus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about10%” would encompass 9-11%.

Unless otherwise indicated, any percentages stated herein are percent byweight.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

Increased amounts of an isoprenoid produced by a genetically alteredyeast may be the result of mutating or modifying one or more enzymeswithin the isoprenoid biosynthesis pathway. For example acetyl-CoAcarboxylase (or “ACCase”), HMG-CoA reductase, squalene epoxidase, andsqualene synthase may be modified or mutated.

In preferred embodiments, the genetically altered yeast expressing amodified enzyme is produced by introducing a mutation in the enzymethrough use of a gene repair oligonucleobase as described herein. Themethod comprises introducing a gene repair oligonucleobase containing aspecific mutation for target gene of interest into a yeast cell by anyof a number of methods well-known in the art (e.g., microcarriers,microfibers, electroporation, microinjection, LiOAc, biolistics,spheroplasting, and/or Agrobacterium (see, for example, McClelland, C.M., Chang, Y. C., and Kwon-Chung, K. J. (2005) Fungal Genetics andBiology 42:904-913) and identifying a cell having the mutated enzyme.

In one aspect of the invention, isoprenoids extracted from a geneticallyaltered yeast disclosed herein are provided. In a related aspect,provided is squalene extracted from a genetically altered yeast asdescribed herein.

In another aspect of the invention, a method of producing isoprenoids,preferably squalene, is provided. In certain embodiments the methodincludes providing a genetically altered yeast as described herein andextracting squalene from the yeast.

In still further embodiments of the above aspects of the invention thereare provided isoprenoids extracted from the above genetically altered ortransgenic yeast.

Types of Yeast.

The compositions and methods as disclosed herein can be based on any ofa number of yeast species or strains. In certain embodiments, the yeastis an oleaginous yeast. For example the yeast may be Cryptococcuscurvatus (for example ATCC 20508), Yarrowia lipolytica (for example ATCC20688 or ATCC 90811), Rhodotorula glutinus (for example ATCC 1078S orATCC 204091), and Rhorosporidium toruloides. The inventors havediscovered that, relative to certain other yeast (such as Yarrowialipolytica), Cryptococcus curvatus and Rhodotorula glutinis grow to veryhigh cell densities on a wide variety of substrates, and produce largeamounts of total lipid under many culture conditions. Accordingly, incertain embodiments Cryptococcus curvatus and Rhodotorula glutinis maybe particularly advantageous for the compositions and methods asdisclosed herein. There are many genetic tools (for example,transformation protocols, selectable markers) that are well developedand specific for Yarrowia lipolytica; as such in some embodimentsYarrowia lipolytica may be particularly advantageous for thecompositions and methods as disclosed herein.

Gene Repair Oligonucleobases

The invention can be practiced with “gene repair oligonucleobases”having the conformations and chemistries as described in detail below.The “gene repair oligonucleobases” of the invention include mixed duplexoligonucleotides, non-nucleotide containing molecules, single strandedoligodeoxynuceotides and other gene repair molecules described in thebelow noted patents and patent publications. The “gene repairoligonucleobases” of the invention have also been described in publishedscientific and patent literature using other names including“recombinagenic oligonucleobases;” “RNA/DNA chimeric oligonucleotides:”“chimeric oligonucleotides;” “mixed duplex oligonucleotides (MDONs);”“RNA DNA oligonucleotides (RDOs);” “gene targeting oligonucleotides;”“genoplasts;” “single stranded modified oligonucleotides;” “Singlestranded oligodeoxynucleotide mutational vectors;” “duplex mutationalvectors;” and “heteroduplex mutational vectors.”

Oligonucleobases having the conformations and chemistries described inU.S. Pat. No. 5,565,350 by Kmiec (Kmiec I) and U.S. Pat. No. 5,731,181by Kmiec (Kmiec II), hereby incorporated by reference, are suitable foruse as “gene repair oligonucleobases” of the invention. The gene repairoligonucleobases in Kmiec I and/or Kmiec II contain two complementarystrands, one of which contains at least one segment of RNA-typenucleotides (an “RNA segment”) that are base paired to DNA-typenucleotides of the other strand.

Kmiec II discloses that purine and pyrimidine base-containingnon-nucleotides can be substituted for nucleotides. Additional generepair molecules that can be used for the present invention aredescribed in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983;5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and inInternational Patent No. PCT/US00/23457; and in International PatentPublication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; andWO 99/40789, which are each hereby incorporated in their entirety.

In one embodiment, the gene repair oligonucleobase is a mixed duplexoligonucleotide in which the RNA-type nucleotides of the mixed duplexoligonucleotide are made RNase resistant by replacing the 2′-hydroxylwith a fluoro, chloro or bromo functionality or by placing a substituenton the 2′-O. Suitable substituents include the substituents taught bythe Kmiec II. Alternative substituents include the substituents taughtby U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught bypatent publications EP 629 387 and EP 679 657 (collectively, the MartinApplications), which are hereby incorporated by reference. As usedherein, a 2′-fluoro, chloro or bromo derivative of a ribonucleotide or aribonucleotide having a 2′-OH substituted with a substituent describedin the Martin Applications or Sproat is termed a “2′-SubstitutedRibonucleotide.” As used herein the term “RNA-type nucleotide” means a2′-hydroxyl or 2′-Substituted Nucleotide that is linked to othernucleotides of a mixed duplex oligonucleotide by an unsubstitutedphosphodiester linkage or any of the non-natural linkages taught byKmiec I or Kmiec II. As used herein the term “deoxyribo-type nucleotide”means a nucleotide having a 2′-H, which can be linked to othernucleotides of a gene repair oligonucleobase by an unsubstitutedphosphodiester linkage or any of the non-natural linkages taught byKmiec I or Kmiec II.

In a particular embodiment of the present invention, the gene repairoligonucleobase is a mixed duplex oligonucleotide that is linked solelyby unsubstituted phosphodiester bonds. In alternative embodiments, thelinkage is by substituted phosphodiesters, phosphodiester derivativesand non-phosphorus-based linkages as taught by Kmiec II. In yet anotherembodiment, each RNA-type nucleotide in the mixed duplex oligonucleotideis a 2′-Substituted Nucleotide. Particular preferred embodiments of2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-propyloxy,2′-allyloxy, 2′-hydroxylethyloxy, 2′-methoxyethyloxy, 2′-fluoropropyloxyand 2′-trifluoropropyloxy substituted ribonucleotides. More preferredembodiments of 2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy,2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In anotherembodiment the mixed duplex oligonucleotide is linked by unsubstitutedphosphodiester bonds.

Although mixed duplex oligonucleotides having only a single type of2′-substituted RNA-type nucleotide are more conveniently synthesized,the methods of the invention can be practiced with mixed duplexoligonucleotides having two or more types of RNA-type nucleotides. Thefunction of an RNA segment may not be affected by an interruption causedby the introduction of a deoxynucleotide between two RNA-typetrinucleotides, accordingly, the term RNA segment encompasses such as“interrupted RNA segment.” An uninterrupted RNA segment is termed acontiguous RNA segment. In an alternative embodiment an RNA segment cancontain alternating RNase-resistant and unsubstituted 2′-OH nucleotides.The mixed duplex oligonucleotides preferably have fewer than 100nucleotides and more preferably fewer than 85 nucleotides, but more than50 nucleotides. The first and second strands are Watson-Crick basepaired. In one embodiment the strands of the mixed duplexoligonucleotide are covalently bonded by a linker, such as a singlestranded hexa, penta or tetranucleotide so that the first and secondstrands are segments of a single oligonucleotide chain having a single3′ and a single 5′ end. The 3′ and 5′ ends can be protected by theaddition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotidesare Watson-Crick paired to adjacent nucleotides. A second hairpin capcan, additionally, be placed at the junction between the first andsecond strands distant from the 3′ and 5′ ends, so that the Watson-Crickpairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologouswith two fragments of the target gene, i.e., have the same sequence asthe target gene. A homologous region contains the nucleotides of an RNAsegment and may contain one or more DNA-type nucleotides of connectingDNA segment and may also contain DNA-type nucleotides that are notwithin the intervening DNA segment. The two regions of homology areseparated by, and each is adjacent to, a region having a sequence thatdiffers from the sequence of the target gene, termed a “heterologousregion.” The heterologous region can contain one, two or threemismatched nucleotides. The mismatched nucleotides can be contiguous oralternatively can be separated by one or two nucleotides that arehomologous with the target gene. Alternatively, the heterologous regioncan also contain an insertion or one, two, three or of five or fewernucleotides. Alternatively, the sequence of the mixed duplexoligonucleotide may differ from the sequence of the target gene only bythe deletion of one, two, three, or five or fewer nucleotides from themixed duplex oligonucleotide. The length and position of theheterologous region is, in this case, deemed to be the length of thedeletion, even though no nucleotides of the mixed duplex oligonucleotideare within the heterologous region. The distance between the fragmentsof the target gene that are complementary to the two homologous regionsis identically the length of the heterologous region when a substitutionor substitutions is intended. When the heterologous region contains aninsertion, the homologous regions are thereby separated in the mixedduplex oligonucleotide farther than their complementary homologousfragments are in the gene, and the converse is applicable when theheterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part ofa homologous region, i.e., a region that is identical in sequence to afragment of the target gene, which segments together preferably containat least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-typenucleotides or yet more preferably 18-22 RNA-type nucleotides or mostpreferably 20 nucleotides. In one embodiment, RNA segments of thehomology regions are separated by and adjacent to, i.e., “connected by”an intervening DNA segment. In one embodiment, each nucleotide of theheterologous region is a nucleotide of the intervening DNA segment. Anintervening DNA segment that contains the heterologous region of a mixedduplex oligonucleotide is termed a “mutator segment.”

In another embodiment of the present invention, the gene repairoligonucleobase is a single stranded oligodeoxynucleotide mutationalvector (SSOMV), which is disclosed in International Patent ApplicationPCT/US00/23457, U.S. Pat. Nos. 6,271,360, 6,479,292, and 7,060,500 whichis incorporated by reference in its entirety. The sequence of the SSOMVis based on the same principles as the mutational vectors described inU.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972,5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in InternationalPublication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; andWO 99/40789. The sequence of the SSOMV contains two regions that arehomologous with the target sequence separated by a region that containsthe desired genetic alteration termed the mutator region. The mutatorregion can have a sequence that is the same length as the sequence thatseparates the homologous regions in the target sequence, but having adifferent sequence. Such a mutator region can cause a substitution.Alternatively, the homologous regions in the SSOMV can be contiguous toeach other, while the regions in the target gene having the samesequence are separated by one, two or more nucleotides. Such a SSOMVcauses a deletion from the target gene of the nucleotides that areabsent from the SSOMV. Lastly, the sequence of the target gene that isidentical to the homologous regions may be adjacent in the target genebut separated by one two or more nucleotides in the sequence of theSSOMV. Such an SSOMV causes an insertion in the sequence of target gene.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked byunmodified phosphodiester bonds except that the 3′ terminal and/or 5′terminal internucleotide linkage or alternatively the two 3′ terminaland/or 5′ terminal internucleotide linkages can be a phosphorothioate orphosphoamidate. As used herein an internucleotide linkage is the linkagebetween nucleotides of the SSOMV and does not include the linkagebetween the 3′ end nucleotide or 5′ end nucleotide and a blockingsubstituent, see supra. In a specific embodiment the length of the SSOMVis between 21 and 55 deoxynucleotides and the lengths of the homologyregions are, accordingly, a total length of at least 20 deoxynucleotidesand at least two homology regions should each have lengths of at least 8deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding orthe non-coding strand of the target gene. When the desired mutation is asubstitution of a single base, it is preferred that both the mutatornucleotide be a pyrimidine. To the extent that is consistent withachieving the desired functional result it is preferred that both themutator nucleotide and the targeted nucleotide in the complementarystrand be pyrimidines. Particularly preferred are SSOMV that encodetransversion mutations, i.e., a C or T mutator nucleotide is mismatched,respectively, with a C or T nucleotide in the complementary strand.

In addition to the oligodeoxynucleotide the SSOMV can contain a 5′blocking substituent that is attached to the 5′ terminal carbons througha linker. The chemistry of the linker is not critical other than itslength, which should preferably be at least 6 atoms long and that thelinker should be flexible. A variety of non-toxic substituents such asbiotin, cholesterol or other steroids or a non-intercalating cationicfluorescent dye can be used. Particularly preferred as reagents to makeSSOMV are the reagents sold as Cy3.™. and Cy5.™. by Glen Research,Sterling Va., which are blocked phosphoroamidites that uponincorporation into an oligonucleotide yield 3,3,3′,3′-tetramethylN,N′-isopropyl substituted indomonocarbocyanine and indodicarbocyaninedyes, respectively. Cy3 is the most preferred. When the indocarbocyanineis N-oxyalkyl substituted it can be conveniently linked to the 5′terminal of the oligodeoxynucleotide through as a phosphodiester with a5′ terminal phosphate. The chemistry of the dye linker between the dyeand the oligodeoxynucleotide is not critical and is chosen for syntheticconvenience. When the commercially available Cy3 phosphoramidite is usedas directed the resulting 5′ modification consists of a blockingsubstituent and linker together which are a N-hydroxypropyl,N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine.

In the preferred embodiment the indocarbocyanine dye is tetrasubstituted at the 3 and 3′ positions of the indole rings. Withoutlimitations as to theory these substitutions prevent the dye from beingan intercalating dye. The identity of the substituents as thesepositions are not critical. The SSOMV can in addition have a 3′ blockingsubstituent. Again the chemistry of the 3′ blocking substituent is notcritical.

Heterologous Expression

In certain embodiments, heterologous expression is used to expressforeign genes or extra copies of endogenous genes in yeast (for example,Yarrowia lipolytica). Heterologous expression in yeast can be preferredusing methods well known in the art. Expression of foreign genes orextra copies of endogenous genes in yeast using heterologous expressionmay involve use of a vector that includes (a) promoter sequences fortranscriptional initiation, (b) terminator sequences for termination oftranscription, and (c) a selectable marker. Heterologous expression andexpression vectors may be as described, for example, in Madzak, C.,Gaillardin, C., and Beckerich, J-M., 2004 Heterologous ProteinExpression and Secretion in the Non-Conventional Yeast Yarrowialipolytica: a review, Journal of Biotechnology 109:63-81. A non-limitinglist of selectable marker genes that may be used includes ura3, lys5,trp1, leu2, ade1, E. coli hph encoding hygromycin resistance, and SUC2from Saccharomyces cerevisiae. A non-limiting list of promoters that maybe used includes pLEU2, pXPR2, pPOX2, pPOT1, pICL1, pG3P, pMTP, pTEF,and pRPS7. A non-limiting list of terminator sequences that may be usedincludes, XPR2t, LIP2t, and PHO5t.

In certain embodiments, one or more of Yarrowia lipolytica LYS1(Genolevures YALI0B15444g), TRP1 (Genolevures YALI0B107667g), and ADE1(Genolevures YALI0E33033g) genes are used as selectable markers.

In certain embodiments an integrative expression vector includes one ormore promoters and/or terminator sequences selected from the groupconsisting of Yarrowia lipolytica glycolytic pathway genes, alkane orglycerol utilization genes, ACC1, HMG1, ERG1, and ERG9.

In certain embodiments of one or both subunits of Yarrowia lipolyticaATP citrate lyase (Genoleveres YALI0D24431g and YALI0E34793g) inYarrowia lipolytica are overexpressed.

Modified Enzymes

The genes encoding enzymes involved in the fatty acid biosynthesispathway and isoprenoid biosynthesis pathway are the preferred targetsfor mutation. In some embodiments the target gene encodes an acyl CoAcarboxylase. In other embodiments the target gene encodes an HMG-CoAreductase. In other embodiments the target gene encodes a squaleneepoxidase. In other embodiments the target gene encodes a squalenesynthase. In certain embodiments the target gene encodes ATP citratelyase. Mutations can be designed that reduce or eliminate the activityof an enzyme, enhance the activity of an enzyme, or that alter theactivity of the enzyme (e.g., change the substrate selectivity).

In wild-type oleaginous yeast, acetyl-CoA is extensively channeled intofatty acid biosynthesis via acetyl-CoA carboxylase (ACCase). Thus inorder to increase the amount of acetyl-CoA available for squalenesynthesis, it is desirable to reduce the enzymatic expression orspecific activity of ACCase. An exemplary gene sequence for ACCase isthe ACC1 gene in Saccharomyces cerevisiae as shown in accession numberZ71631. Accordingly in certain embodiments reduced intracellularactivities of ACCase, the enzyme at the branch point between mevalonatebiosynthesis and triglyceride biosynthesis will decrease the amount ofacetyl-CoA partitioned for oil synthesis thereby increasing itsavailability to the isoprene pathway.

HMG-CoA reductase activity is the rate-limiting enzyme for isoprenebiosynthesis. Exemplary gene sequences for HMG-CoA reductase include theHMG1 and HMG1 genes in Saccharomyces cerevisiae as shown in accessionnumbers NC_001145 and NC_001144, respectfully. Accordingly. in certainembodiments HMG-CoA reductase activity will be increased by modifyingthe HMGR gene to increase transcription, stabilize the resultantprotein, and/or reduce product feedback inhibition.

Decreasing ACCase activity and/or increasing HMG-CoA reductase activityin a yeast can create a core isoprenoid production organism capable ofproducing a number of related isoprenoid products by the manipulation ofsubsequent enzymes in the pathway.

Squalene epoxidase catalyzes the first committed step of sterolbiosynthesis. An exemplary gene sequence for Squalene epoxidase is theERG1 gene in Saccharomyces cerevisiae as shown in accession numberNC_001139. Accordingly, in certain embodiments squalene epoxidaseactivity and/or expression will be attenuated in a yeast, for example bycatalytically important residues in the enzyme's amino acid sequence.

Squalene synthase catalyzes the synthesis of squalene by condensing twoc15 isoprene precursors (farnesyl diphosphate (FPP)). An exemplary genesequence for squalene synthase is the ERG9 gene in Saccharomycescerevisiae as shown in accession number NC_001140. Accordingly, incertain embodiments squalene synthase activity and/or expression will beincreased in a yeast.

ATP citrate lyase (E.C. 4.1.3.8) catalytically cleaves citrate toproduce acetyl CoA and oxaloacetate. Acetyl CoA can be used by ACCasefor fatty acid biosynthesis or by acetyl CoA acetyl transferase for theproduction of isoprenes and derivatives such as squalene.

The result of the metabolic changes will be to channel carbon fromacetyl-CoA to squalene, and attenuate major competitive pathways forthis carbon stream, resulting in a significant increase of squaleneproduced.

Delivery of Gene Repair Oligonucleobases into Yeast Cells

Any commonly known method can be used in the methods of the presentinvention to transform a yeast cell with a gene repair oligonucleobases.Exemplary methods include the use of microcarriers or microfibers,microinjection, by electroporation, LiOAc, biolistics, spheroplasting,and/or Agrobacterium (see, for example, McClelland, C. M., Chang, Y. C.,and Kwon-Chung, K. J. (2005) Fungal Genetics and Biology 42:904-913).

In some embodiments, metallic microcarriers (microspheres) are used tointroduce large fragments of DNA into yeast cells having cell walls byprojectile penetration (biolistic delivery) and is well known to thoseskilled in the relevant art. General techniques for selectingmicrocarriers and devices for projecting them are described in U.S. Pat.Nos. 4,945,050; 5,100,792 and 5,204,253.

Specific conditions for using microcarriers in the methods of thepresent invention are described in International Publication WO99/07865, US09/129,298. For example, ice cold microcarriers (60 mg/mL),mixed duplex oligonucleotide (60 mg/mL), 2.5 M CaCl₂ and 0.1 Mspermidine are added in that order; the mixture gently agitated, e.g.,by vortexing, for 10 minutes and let stand at room temperature for 10minutes, whereupon the microcarriers are diluted in 5 volumes ofethanol, centrifuged and resuspended in 100% ethanol. Exemplaryconcentrations of the components in the adhering solution include 8-10μg/μL microcarriers, 14-17 μg/μL mixed duplex oligonucleotide, 1.1-1.4 MCaCl₂ and 18-22 mM spermidine. In one example, the componentconcentrations are 8 μg/L microcarriers, 16.5 μg,/L mixed duplexoligonucleotide, 1.3 M CaCl₂ and 21 mM spermidine.

Gene repair oligonucleobases can also be introduced into yeast cells forthe practice of the present invention using microfibers to penetrate thecell wall and cell membrane. U.S. Pat. No. 5,302,523 to Coffee et al.describes the use of 30×0.5 μm and 10×0.3 μm silicon carbide fibers tofacilitate transformation of suspension maize cultures of Black MexicanSweet. Any mechanical technique that can be used to introduce DNA fortransformation of a yeast cell using microfibers can be used to delivergene repair oligonucleobases.

One example of microfiber delivery of a gene repair oligonucleobase isas follows. Sterile microfibers (2 μg) are suspended in 150 μL of yeastgrowth medium containing about 10 μg of a mixed duplex oligonucleotide.A yeast culture is allowed to settle and equal volumes of packed cellsand the sterile fiber/nucleotide suspension are vortexed for 10 minutesand plated. Selective media are applied immediately or with a delay ofup to about 120 hours as is appropriate for the particular trait.

In an alternative embodiment, the gene repair oligonucleobases can bedelivered to the yeast cell by electroporation of a protoplast derivedfrom yeast cells. The protoplasts are formed by enzymatic treatment of ayeast cells, according to techniques well known to those skilled in theart. (See, e.g., Gallois et al., 1996, in Methods in Molecular Biology55:89-107, Humana Press, Totowa, N.J.; Kipp et al., 1999, in Methods inMolecular Biology 133:213-221, Humana Press, Totowa, N.J.) Theprotoplasts need not be cultured in growth media prior toelectroporation. Illustrative conditions for electroporation are 3×10⁵protoplasts in a total volume of 0.3 mL with a concentration of generepair oligonucleobase of between 0.6-4 μg/mL.

In yet another alternative embodiment, the gene repair oligonucleobasecan be delivered to the yeast cell by whiskers or microinjection of theyeast cell. The so-called whiskers technique is performed essentially asdescribed in Frame et al., 1994, Plant J. 6:941-948. The gene repairoligonucleobase is added to the whiskers and used to transform the yeastcells. The gene repair oligonucleobase may be co-incubated with plasmidscomprising sequences encoding proteins capable of forming recombinaseand/or gene repair complexes in yeast cells such that gene repair iscatalyzed between the oligonucleotide and the target sequence in thetarget gene.

Selection of Yeast Having the Desired Modified Enzyme

Yeast expressing the modified enzyme can be identified through any of anumber of means. In one method, a co-conversion strategy using generepair oligonucleobases (GRONs) to target both a selectable conversion(i.e., a marker) and a non-selectable conversion (e.g., a target gene ofinterest) in the same experiment. In this way, the cells to which GRONswere not delivered or were unable to transmit the conversions specifiedby the RON would be eliminated. Since delivery of GRONs targetingunrelated genes is not expected to be selective. at some frequency, acolony with a successfully selected conversion would also be expected tohave a conversion in one of the other targeted genes. Conversion eventswould be resolved by single nucleotide polymorphism (SNP) analysis.

Thus, genomic DNA is extracted from from yeast and screening of theindividual DNA samples using a SNP detection technology, eg.allele-specific Polymerase Chain Reaction (ASPCR), for each target. Toindependently confirm the sequence change in positive yeast. theappropriate region of the target gene may be PCR amplified and theresulting amplicon either sequenced directly or cloned and multipleinserts sequenced.

Alternatively, the incorporation of the mutation into the gene ofinterest can be identified by any of a number of molecular biologytechniques designed to detect single nucleotide mutations in extractednucleic acid (e.g., amplification methods such as PCR and singlenucleotide primer extension analysis). Larger mutations can be detectedby amplification and sequencing of the region of the target gene to bemutated.

Alternatively, yeast or yeast cells containing the modified enzyme canbe identified by, for example, analysis of the composition ofisoprenoids produced by the yeast. Thus, the yeast can be grown and oilsextracted and analyzed using methods known in the art (e.g., gaschromatography).

EXAMPLES Example 1 Cryptococcus curvatus and Rhodotorula glutinisTransformation Systems

To create a Cryptococcus curvatus (ATCC strain 20508) and Rhodotorulaglutinis (ATCC strains 10788 and 204091) transformation system, a KANMXexpression cassette (promoter-gene-terminator) which confers kanamycinresistance to S. cerevisiae is used as a selectable marker to convertthe strains from Kanamycin sensitivity to resistance (See e.g. Baudin,A., et al. (1993) Nucleic Acids Research (21) 3329-3330). The strainsare transformed with the expression cassette alone, as well as KANMXligated to restriction fragments of a plasmid reported in R. glutinus(See e.g Oloke, J. K., and Glick, B. R. (2006) African Journal ofBiotechnology 5(4):327-332) containing DNA origins of replication. DNAis introduced into C. curvatus and R. glutinis by electroporation,LiOAc, biolistics, spheroplasting, and/or Agrobacterium (McClelland, C.M., Chang, Y. C. and Kwon-Chung K. J. (2005) Fungal Genetics and Biology42:904-913).

Example 2 Selectable Markers

To generate uracil auxotrophic mutants in Cryptococcus curvatus andRhodotorula glutinis, cells were grown in minimal media containinganti-metabolite 5-fluoroorotic acid to select for resistant mutants withlesions in the ura3 or ura5 genes. 33 stable 5-FOA^(R) colonies ofCryptococcus curvatus and 20 stable 5-FOA^(R) colonies of Rhodotorulaglutinis were banked. Wild type URA3 genes from both Cryptococcuscurvatus and Rhodotorula glutinis are cloned and the mutant ura3 genesin the 5-FOA^(R) isolates are sequenced.

Other auxotrophic markers are cloned by functional complementation inSaccharomyces cerevisiae (See Ho, Y. R., and Chang, M. C. (1988) ChineseJournal of Microbiology and Immunology 21(1):1-8). Genomic and/or cDNAlibraries are constructed from Cryptococcus curvatus and Rhodotorulaglutitnis for ligation into a uracil-selectable Saccharomyces expressionvector for transformation into strain YPH500 (MATα ura3-52 lys2-80Vade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) to select for lysine, adenine,tryptophan, histidine, and leucine prototrophs. From these prototrophs,the corresponding genes for LYS2, ADE2, TRP1, HIS3, and LEU2 aresequenced from the genomic or cDNA insert.

Example 2 Gene Manipulation in Yeast Using RTDS Technology

The alleles of the leu2, lys5 and ura3 genes from Yarrowia lipolyticastrain ATCC 90811 (leu2-35 lys5-12 ura3-18 XPR2B) were cloned by PCR andtheir sequences compared to the wild type alleles to identifydifferences.

For ura3, differences were found at positions 1365 (A→G mutation.resulting in a silent change of AAA→AAG coding for lysine), 1503 (AAGAAextra sequences in ATCC 90811 which results in a frame change, but whichcomes back in frame at 1511 resulting in 7 additional amino acids, afterwhich the sequence continues as the YL URA3 in GenBank), 1511 (extra Tin ATCC 90811), and 1978 (C→T mutation, leading to a stop mutationtruncating the protein 24 amino acids short of the carboxy terminus). AGRON oligonucleotide was designed to restore prototrophy by convertingSTOP(TGA)->R (CGA) to yield 264R based on YlUra3-YLU40564 amino acidnumbering. The GRONs used are YlUra31264/C/40/5′Cy3/3′idC, which has thesequence VCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGGH, andYlUra31264/NC/40,5′Cy3/3′idC, which has the sequenceVCCTCCTCAATAGGATCTCGGTTCTGGCCGTACAGACCTGH, where V=CY3; H=3DMT dC CPG.10, 30, and 50 μg of each of the GRONs were transformed into Yarrowialipolytica strain ATCC 90811 using a Lithium acetate-based method, andplated onto ura-2% glucose. A total of 82 ura+ colonies were obtainedwith the GRON designed using the coding strand and 6 colonies with theGRON designed using the non-coding strand, demonstrating the strand biascommon in transforming with gap-repair oligonucleotides. Sequencing of18 of the coding-strand transformants demonstrated the intended changein 17 of the clones.

For LEU2 differences were found at positions 1710 (extra C absentleading to a frame shift and premature protein termination); 1896 (extraT); 2036 (T→A mutation, located after the stop codon); 2177 (extra T inmissing, located after stop codon),

For LEU2 differences were found at positions 1092 (G→A TCG→TCA, aconservative substitution (Serine)); 1278 (G→A CAG→CAA, a conservativesubstitution (Glutamine)); 1279 (G→A GGT→ATT, changing V→I).

Accordingly, the mutations can be used for various purposes, for exampleto convert prototrophic yeast to become auxotrophic and vice versa,

A similar strategy for demonstrating the effectiveness of RTDStechnology in Cryptococcus curvatus and Rhodotorula glutinis isperformed as described for Yarrowia lipolytica in which ura3 mutationsare corrected to restore prototrophy.

Example 3 Cloning of Target Genes

The sequences for ACCase, HMGR, squalene synthase and squaleneepoxidase, available in the NCBI database from Saccharomyces and otheryeasts, are used as a source of PCR primers and the corresponding genesare cloned from Cryptococcus curvatus and Rhodotorula glutinis alongwith their corresponding regulatory regions (promoters, terminators). Toidentify ‘up’ and ‘down’ promoter mutations that increase or decreasetranscription, respectively, the promoters for these four genes arecloned with a relatively error-prone DNA polymerase to generate pointmutations in the promoters, and these fragments are cloned into plasmidsfused with Green Fluorescent Protein (GFP) or beta-galactosidasereporter genes for testing in vitro in S. cerevisiae or E. coli.Promoter “up” mutations are reintroduced into the HMGR and squalenesynthase genomic sequences by RTDS, while “down” promoter mutations arebeing made in the genomic ACCase and squalene epoxidase sequences. Thepromoters from essential genes (e.g. CAPDH, actin) in R. glutinis and C.curvatus are cloned for use in heterologous gene expression. Primers forPCR cloning are designed from homology to these genes in S. cerevisiae.

Example 4 Manipulation of Target Genes for Increased Squalene Production

ACCase. The number of copies of the ACCase gene is determined in R.glutinis and C. curvatus and other yeasts. RTDS is utilized to reduceACCase expression by introducing stop codons immediately after thetranslational start site in any extra copies. A correlation betweenACCase mRNA levels and ACCase enzymatic specific activity wasdemonstrated in Saccharomyces cerevisiae.

Squalene Epoxidase. Similarly, an increase in squalene accumulation inS. cerevisiae has been achieved by disruption of one copy of thesqualene epoxidase in the diploid. Kamimura, N., Hidaka, M., Masaki, H.,and Uozumi, T. (1994) Appl. Microb. Biotech. 42: 353-357. The number ofcopies of squalene epoxidase in R. glutinis and C. curvatus and otheryeasts is determined, and RTDS is used to create or insert a stop codonimmediately after the translational start site in extra copies beyondthe first one.

HMGR. Both Saccharomyces cerevisiae and mammalian HMGR enzymes containamino acid sequences in their linker regions which are present in manyshort-lived proteins that are subject to rapid intracellular turnover ineukaryotes (see Rogers, S., Wells, R., and Rechsteiner, M. (1986)Science 234: 364-368; and Chun, K. T., and Simoni, R. D. (1991) J. Biol.Chem. 267(6): 4236-4246). Similar sequences, if present, are identifiedin the HMGR genes in R. glutinis and C. curvatus, and eliminated usingRTDS to reduce HMGR protein turnover. Such similar sequences have beenfound in the S. cerevisiae squalene synthase gene, and it is alsodetermined if such sequences are present in the squalene synthase genesin R. glutinis and C. curvatus. The sequences, if present in R. glutinisand C. curvatus squalene synthase, are also eliminated using RTDS toreduce protein turnover.

HMGR in S. cerevisiae comprises two highly conserved domains, of whichthe N-terminal 552 amino acids are responsible for membrane association.Overexpression of the truncated HMG1 protein containing only theC-terminal catalytic portion led a 40-fold increase of HMG-CoA activityin S. cerevisiae with an increased accumulation of squalene to 5.5% ofdry matter (Polakowski, T., Stahl, U., and Lang, C. (1998) Appl.Microbiol. Biotech. 49:66-71). It is determined if R. glutinis and C.curvatus HMGR proteins have a similar structure, and, if so, RTDS isused to express only the soluble catalytic domain.

The protein structure and DNA sequence of HMGR is highly conservedbetween eukaryotes from fungi to mammals, with a membrane-associatedN-terminal domain and catalytic C-terminal domain. The boundary betweenthe two domains can be mapped to a region of amino acids 500-600 in theYarrowia lipolytica HMG1 gene (Genelouvres Yarrowia lipolyticaYALI0E04807g) where the hydrophobicity plot transitions from hydrophobicto hydrophilic. Resides 548 and 544 are chosen from evaluation of thehydrophobicity plot of Yarrowia lipolytica HMG1, and its homology to theN-termini of the truncated Saccharomyces cerevisiae (Donald, K. A. G.,et al, 1997. Appl. Environ. Micro. 63(9): 3341-3344) and Candida utilis(Shimada, H. et al, 1998. Appl. Environ. Micro. 64(7):2676-2680)proteins. Accordingly, in one example, amino acids 548-1000 of theC-terminal domain of Yarrowia lipolytica HMG1 I is expressed; in asecond example amino acids 544-1000 of the C-terminal domain of Yarrowialipolytica HMG1 I is expressed. In related examples, amino acids543-1000 of the C-terminal domain of Yarrowia lipolytica HMG1 I isexpressed; or amino acids 545-1000 of the C-terminal domains of Yarrowialipolytica HMG1 I is expressed, or amino acids 546-1000 of theC-terminal domains of Yarrowia lipolytica HMG1 I is expressed; or aminoacids 547-1000 of the C-terminal domains of Yarrowia lipolytica HMG1 Iis expressed; or amino acids 549-1000 of the C-terminal domains ofYarrowia lipolytica HMG1 I is expressed.

In Syrian hamsters, activity of the HMGR catalytic domain isdown-modulated by phosphorylation by an AMP-dependent kinase (Omkumar,R. V., Darnay, B. G., and Rodwell, V. W. (1994) J. Biol. Chem.269:6810-6814), and a similar mode of regulation has been described inS. cerevisiae. It is determined if the HMGR proteins in R. glutinis, C.curvatus and other yeasts are similarly regulated, and if so, RTDS isemployed to eliminate the phosphorylation site.

Squalene synthase. Squalene synthase in mammalian systems isco-ordinately regulated on the transcriptional level along with HMG-CoAsynthase and farnesyl diphosphate synthase by SREBPs (sterol regulatoryelement binding proteins) (Szkopinsda, A., Swiezewska, E., and Karst, F(2000) Biochem. Biophys. Res. Comm. 267:473-477). SREBPs exist in threeforms, of which one binds the squalene synthase promoter. It isdetermined if such transcription factors and/or binding sites arepresent on the squalene synthase promoter in R. glutinis, C. curvatusand other yeasts, and, if present, RTDS is used to make changes to suchtranscription factors and/or binding sites that enhance transcription ofsqualene synthase.

Example 5 Growth Conditions for Cryptococcus curvatus

Cryptococcus curvatus growth was evaluated to determine the best carbonsources to maximize its cell mass in culture. In a Yeast Extract-basedrich media (10 g/L yeast extract, 20 g/L peptone), C. curvatus grew wellin 2-20% w/v glucose, achieving a maximal level of 55 g/L cell dryweight (CDW) at 16% w/v glucose and above after 4 days. Similarly, C.curvatus grew in the same media with 3-12% w/v glycerol, achieving a CDWof 40 g/L in 12% w/v glycerol after 5 days. C. curvatus was also grownin Biodiesel glycerol (Imperial Western Products, Coachella, Calif.) upto 3.5% w/v, resulting in 23 g/L CDW.

Example 6 Environmental Manipulation of Target Genes for IncreasedSqualene Production

Environmental manipulations are tested to increase the net yield ofsqualene. These include (a) inhibiting ACCase expression and/or activitywith oleic acid, olive or other vegetable oil(s), inositol, choline,soraphen, fluazifop, and clethodim or other ACCase inhibitingherbicides, (b) inhibiting squalene epoxidase expression and/or activitywith terbinafine, tolnaftate, and ergosterol or other squalene epoxidaseinhibiting fungicides, (c) manipulating the C/N ratio in glycerol-basedmedia (in the starting media or by add-ins), (d) varying the nitrogensource in the media (organic vs. inorganic vs. simple/complex), (e)varying carbon addition regimes (e.g. batch vs. feeding), (f) examiningthe effect of depleting nutrients other than carbon source, (g) varyingthe carbon source to include mixtures of sugars, sugar alcohols,alcohols, polyalcohols, and organic acids, (h) selecting for growth onHMGR-inhibitory compounds such as lovastatin or other statin-typeinhibitors, and (i) selecting for high oil production in culture usinglipophillic dyes or stains and/or by analyzing for extractable lipidsusing, for example, gravimetric or gas chromatographic methods.

For example, Yarrowia lipolytica ATCC 90904 was cultivated in highCarbon/Nitrogen ratio media (C/N=420, Li, Y-H., Liu, B., Zhao, Z-B., andBai, F-W. 2006 “Optimized Culture Medium and Fermentation Conditions forLipid Production by Rhodosporidium toruloides” Chinese Journal ofBiotechnology 22(4): 650-656) (hereinafter “CYM001 Media”) supplementedwith 0 to 50 μg/ml terbinafine at 30° C., 300 rpm for 120 h.Concentrations of 12.5 μg/ml or higher of terbinafine resulted in up to18.5% of total lipid as squalene.

In another example, Yarrowia lipolytica ATCC 90904 was cultivated inCYM001 media supplemented with 0 to 50 μg/ml Oleic acid at 30° C., 300rpm for 120 h. Supplementation with 10 μl/ml Oleic acid was found toimprove lipid accumulation to 63.3% lipid/CDW (cell dry weight).

In a further example, Yarrowia lipolytica ATCC 90904 was cultivated inCYM001 media supplemented with 0 to 200 μM clethodim at 30° C., 300 rpmfor 120 h. Supplementation of 200 μM clethodim resulted in a 60-foldincrease in the yield (mg) of squalene per 60-ml flask.

Increased oxygen has been shown to cause the differential regulation ofHMG1 and HMG2 in S. cerevisiae, resulting in rapid degradation of HMG2and increased expression of HMG1 under aerobic conditions (Casey, W. M.,Keesler, G. A., Parks, L. W. (1992) J. Bact. 174:7283-7288). It isdetermined if the number of HMGR genes in our oleaginous yeasts isaffected by oxygen and, if so, their expression and activity ismanipulated in the fermenter by altering oxygen levels.

Starting with “CYM001 Media” (Li, Y-H., Liu. B., Zhao, Z-B., and Bai,F-W. (2006) Chinese Journal of Biotechnology 22(4):650-656), variouscomponents and concentrations of components are changed (including theaddition of new components) to improve cell growth, percent total lipidcontent/unit mass of cells, and percent squalene/total lipid. Mediacomponents that are evaluated include: carbon sources: glycerol,glucose, nitrogen sources: ammonium compounds, nitrates, amino acids,mineral salts: potassium, magnesium, sodium. iron, manganese, zinc,calcium, copper, yeast extract, lipid precursors and lipid synthesisaffectors: terbinafine, clethodim, oleic acid, palmitoleic acid,linoleic acid, linolenic acid and antifoaming agents. Other factors thatare evaluated include: percent inoculum, elapsed fermentation time,temperature, pH, back pressure, dissolved oxygen (DO), feed composition,feed strategy and agitation strategy.

Example 7 Strain Selection

Traditional strain selection methods are used in oleaginous yeasts toincrease their net squalene productivity. Strains mutagenized by UV,nitrosoguanidine, or ethane methyl sulfonate are screened and/orselected for increased squalene accumulation. Strains are also subjectedto iterative selection pressure, such as repeated passage on YEP (15 g/Lyeast extract, 5 g/L peptone) media containing 3% glycerol or mediacontaining lovastatin and other known HMGR inhibitors. Strains are alsosubjected to repeated passage on CYM001 Media containing varying amountsof glycerol and/or glucose or media containing lovastatin and/or otherknown HMGR inhibitors, and/or squalene synthase inhibitors to obtainspontaneous mutants with increased HMGR and/or squalene synthaseactivity. Such mutations may be in HMGR, squalene synthase, or othergenes (“secondary site mutations”).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

That which is claimed is:
 1. A method of producing squalene by agenetically altered yeast, said method comprising increasing activity orexpression of squalene synthase by a yeast cell by introducing into theyeast cell (i) one or more mutations into a squalene synthase gene ofthe yeast using a gene repair oligonucleobase or (ii) an exogenoussqualene synthase gene, thereby genetically altering the yeast; andselecting a yeast cell expressing squalene synthase having the one ormore mutations, or selecting a yeast cell expressing the exogenoussqualene synthase gene, wherein said genetically altered yeast producesincreased quantities of squalene as compared to yeast expressingsqualene synthase which lacks the one or more mutations or yeast lackingthe exogenous squalene synthase gene.
 2. The method of claim 1, whereinsaid yeast is an oleaginous yeast.
 3. The method of claim 1, whereinsaid yeast is selected from the group consisting of Lipomyces lipofer,L. starkeyi, L. tetrasporus, Candida lipolytica, C. diddensiae, C.paralipolytica, C. curvata, Cryptococcus albidus, Cryptococcuslaurentii, Geotrichum candidum, Rhodotorula graminus, Trichosporonpullulans, Rhodosporidium toruloides, Rhodotorula glutinus, Rhodotorulagracilis, and Yarrowia lipolytica.
 4. The method of claim 1, whereinsaid yeast is not Yarrowia lipolytica.
 5. The method of claim 1, whereinsaid genetically altered yeast produces at least 2-fold more squalene incomparison to yeast expressing squalene synthase which lacks the one ormore mutations or yeast lacking the exogenous squalene synthase gene. 6.The method of claim 1, further comprising extracting squalene from thegenetically altered yeast.